Fluorescence Detector for Microfluidic Diagnostic System

ABSTRACT

The present technology provides for an fluorescent detector that is configured to detect light emitted for a probe characteristic of a polynucleotide. The polynucleotide is undergoing amplification in a microfluidic channel with which the detector is in optical communication. The detector is configured to detect minute quantities of polynucleotide, such as would be contained in a microfluidic volume. The detector can also be multiplexed to permit multiple concurrent measurements on multiple polynucleotides concurrently.

CLAIM OF PRIORITY

The instant application claims the benefit of priority to U.S.provisional applications having Ser. Nos. 60/859,284, filed Nov. 14,2006, and 60/959,437, filed Jul. 13, 2007, the specifications of both ofwhich are incorporated herein by reference in their entireties. Theinstant application is also a continuation-in-part of U.S. patentapplication Ser. No. 11/728,964, filed Mar. 26, 2007, the specificationof which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The technology described herein generally relates to systems fordetecting polynucleotides in samples, particularly from biologicalsamples. The technology more particularly relates to microfluidicsystems that carry out PCR on nucleotides of interest withinmicrofluidic channels, and detect those nucleotides.

BACKGROUND

The medical diagnostics industry is a critical element of today'shealthcare infrastructure. At present, however, diagnostic analyses nomatter how routine have become a bottleneck in patient care. There areseveral reasons for this. First, many diagnostic analyses can only bedone with highly specialist equipment that is both expensive and onlyoperable by trained clinicians. Such equipment is found in only a fewlocations—often just one in any given urban area. This means that mosthospitals are required to send out samples for analyses to theselocations, thereby incurring shipping costs and transportation delays,and possibly even sample loss. Second, the equipment in question istypically not available ‘on-demand’ but instead runs in batches, therebydelaying the processing time for many samples because they must wait fora machine to fill up before they can be run.

Understanding that sample flow breaks down into several key steps, itwould be desirable to consider ways to automate as many of these aspossible. For example, a biological sample, once extracted from apatient, must be put in a form suitable for a processing regime thattypically involves using PCR to amplify a vector of interest. Onceamplified, the presence of a nucleotide of interest from the sampleneeds to be determined unambiguously. Sample preparation is a processthat is susceptible to automation but is also relatively routinelycarried out in almost any location. By contrast, steps such as PCR andnucleotide detection have customarily only been within the compass ofspecially trained individuals having access to specialist equipment.

There is a need for a method and apparatus of carrying out PCR anddetection on prepared biological samples, and preferably with highthroughput. In particular there is a need for an easy-to-use device thatcan deliver a diagnostic result on several samples in a short time.

The discussion of the background to the technology herein is included toexplain the context of the technology. This is not to be taken as anadmission that any of the material referred to was published, known, orpart of the common general knowledge as at the priority date of any ofthe claims.

Throughout the description and claims of the specification the word“comprise” and variations thereof, such as “comprising” and “comprises”,is not intended to exclude other additives, components, integers orsteps.

SUMMARY

The present technology addresses systems for detecting polynucleotidesin samples, particularly from biological samples. In particular, thetechnology relates to microfluidic systems that carry out PCR onnucleotides of interest within microfluidic channels, and detect thosenucleotides.

The present technology provides for a fluorescent detector, comprising:a LED emitting light of a specified color that excites a probeassociated with one or more polynucleotides contained within amicrofluidic channel; and a photodiode configured to collect emittedlight of the specified color, wherein the photodiode is connected to apre-amplifier circuit having a time-constant of less than about 1 s.

A diagnostic apparatus, comprising: one or more microfluidic channelsconfigured to amplify one or more polynucleotides; and one or morefluorescence detectors configured to detect presence of the one or morepolynucleotides in the one or more channels by detecting fluorescentlight emitted from a probe associated with the one or morepolynucleotides, wherein the one or more detectors each comprise: afirst LED emitting light of a first color; a second LED emitting lightof a second color; a first photodiode configured to collect emittedlight of the first color; a second photodiode configured to collectemitted light of the second color; and wherein the first and secondphotodiodes are each connected to a pre-amplifier circuit having atime-constant of less than of about 1 second. In certain embodiments,the time constant is 50-100 ms.

In certain other embodiments, the pre-amplifier circuit furthercomprises a resistor having a resistance in excess of 0.5 GΩ.

The detector can be configured to detect fluorescence from one or moremicrofluidic channels in a removable microfluidic cartridge, such asdisposed within a receiving bay in the apparatus.

The technology further provides for a diagnostic apparatus, comprising:one or more microfluidic channels configured to amplify one or morepolynucleotides; and one or more fluorescence detectors configured todetect presence of the one or more polynucleotides in the one or morechannels by detecting fluorescent light emitted from a probe associatedwith the one or more polynucleotides, wherein the one or more detectorseach comprise: a LED emitting light of a specified color that excitesthe probe; a photodiode configured to collect emitted light of thespecified color; and wherein the photodiode is connected to apre-amplifier circuit having a time-constant of less than about 1 s.

The technology further provides for a diagnostic apparatus, comprising:one or more microfluidic channels configured to amplify one or morepolynucleotides; and one or more fluorescence detectors configured todetect presence of the one or more polynucleotides in the one or morechannels by detecting fluorescent light emitted from a probe associatedwith the one or more polynucleotides, wherein the one or more detectorseach comprise: a first LED emitting light of a first color; a second LEDemitting light of a second color; a first photodiode configured tocollect emitted light of the first color; a second photodiode configuredto collect emitted light of the second color; and wherein the first andsecond photodiodes are each connected to a pre-amplifier circuit havinga Gain of about 10⁹.

The details of one or more embodiments of the technology are set forthin the accompanying drawings and further description herein. Otherfeatures, objects, and advantages of the technology will be apparentfrom the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cross-section of a pipetting head, a detector, and acartridge in position in a microfluidic apparatus.

FIG. 2 shows a cross-sectional view of an exemplary detector, invertedrelative to other views herein;

FIG. 3 shows a cutaway view of an exemplary detector in a read-head;

FIGS. 4A and 4B show perspective and cross-sectional views respectivelyof a detector in a read-head;

FIG. 5 shows an exterior view of an exemplary multiplexed read-head withan array of detectors therein;

FIG. 6 shows a cutaway view of an exemplary multiplexed read-head, as inFIG. 5;

FIG. 7 shows exemplary pre-amplifier circuitry for a fluorescencedetector;

FIG. 8A shows effects of aperturing on fluorescence intensity; FIG. 8Bshows a detector in cross section with an exemplary aperture;

FIG. 9A shows an exemplary multi-lane cartridge;

FIG. 9B shows a portion of an exemplary multi-lane cartridge;

FIG. 10 shows a plan of microfluidic circuitry and inlets in anexemplary multi-lane cartridge.

FIG. 11 shows an exemplary microfluidic network in a lane of amulti-lane cartridge;

FIG. 12 shows an exemplary highly-multiplexed microfluidic cartridge;

FIG. 13 shows an exemplary highly-multiplexed microfluidic cartridge;

FIG. 14 shows a radially configured highly multiplexed microfluidiccartridge.

FIG. 15 shows a cross-section of a microfluidic cartridge, when incontact with a heater substrate;

FIGS. 16A and 16B show a plan view of heater circuitry adjacent to a PCRreaction chamber; FIG. 16C shows an overlay of an array of heaterelements on an exemplary multi-lane microfluidic cartridge, whereinvarious microfluidic networks are visible;

FIG. 17 shows an exemplary layout for electronics and softwarecomponents, as further described herein;

FIG. 18 shows an exemplary apparatus, a microfluidic cartridge, and aread head, as further described herein;

FIG. 19 shows an exploded view of an exemplary apparatus;

FIGS. 20A and 20B show an exemplary apparatus having a detector mountedin a sliding lid;

FIGS. 21A-21C show a force member;

FIGS. 22A-22 D show a force member associated with a detector;

FIG. 23 shows a block diagram of exemplary electronic circuitry inconjunction with a detector as described herein;

FIG. 24 shows a cross-section of a detector;

FIG. 25 shows an interface for exemplary software;

FIG. 26 shows a cross-section of a scanning read-head;

FIG. 27 shows cut-away views of a scanning readhead;

FIGS. 28A and 28B show a microfluidic cartridge aligned to an apertureplate;

FIG. 29 shows a perspective view of a scanning read head; and

FIG. 30 shows cutaway and cross-section views of a read head.

DETAILED DESCRIPTION Overview

One aspect of the present technology relates to a fluorescence detectionsystem for use with a microfluidic-based diagnostic system. Inparticular, the detection system described herein is configured todetect presence of a nucleotide amplified by, e.g., a polymerase chainreaction (PCR), in a microfluidic channel. It is to be understood that,unless specifically made clear to the contrary, where the term PCR isused herein, any variant of PCR including but not limited to real-timeand quantitative, and any other form of polynucleotide amplification isintended to be encompassed.

As further described herein, a microfluidic channel, within whichpresence of an analyte is detected by the detection system, is typicallya chamber or a reactor, such as a PCR reactor, wherein a sample issubjected to a temperature protocol that causes one or more reactions tooccur.

Channels of a microfluidic network in a lane of a microfluidicsubstrate, such as in a cartridge, typically have at least onesub-millimeter cross-sectional dimension. For example, channels of sucha network may have a width and/or a depth of about 1 mm or less (e.g.,about 750 microns or less, about 500 microns, or less, about 250 micronsor less).

By microfluidic, as used herein, is meant that volumes of sample, and/orreagent, and/or amplified polynucleotide are from about 0.1 μl to about999 μl, such as from 1-100 μl, or from 2-μl. Similarly, as applied to acartridge, the term microfluidic means that various components andchannels of the cartridge, as further described herein, are configuredto accept, and/or retain, and/or facilitate passage of microfluidicvolumes of sample, reagent, or amplified polynucleotide.

Furthermore, the detection system can be configured to simultaneouslydetect presence of several nucleotides, distributed amongst severalmicrofluidic channels. The microfluidic channel or channels may be in,for example, a microfluidic substrate, such as found in a microfluidiccartridge, with which the detector is in communication. In particular,the detection system is configured to detect very weak signals as arecharacteristic of samples having very small effective amounts of theanalyte (e.g., polynucleotide) whose presence is being determined. Thedetector is typically mounted within an apparatus that controls theprogress of the amplification reaction in the one or more microfluidicchannels, such as by controlled selective application of localized heatto the one or more channels, wherein the apparatus is also typicallyable to accept user instructions as input, and to provide the result ofdetection as an output. Other characteristics of such an apparatus aredescribed further herein.

Embodiments of the optical system described herein are configured tomeasure fluorescence from a real-time PCR reaction but it would beunderstood that the principles involved could be transferred tomonitoring other reactions on the same or similar scales. The processamplifies a single copy of target DNA into millions or billions ofcopies depending on the number of PCR cycles performed in the reaction.As most of the real-time PCR reaction systems such as Taqman or Scorpionchemistries involve one to one correspondence between the number oftarget DNA copies and the number of fluorescent probes, the amount offluorescence emanating from a reaction volume should be detectable by asensitive fluorescent detection system, as is described herein. Assumingthe PCR reaction is 100% efficient, the sensitivity of the PCR system isproportional to the detection volume seen by the photodetector. However,as DNA molecules will diffuse approximately a millimeter over a time of14-20 minutes (the typical total time required to perform 45 cycles ofPCR according to methods described herein) and the probe molecules maydiffuse an even greater distance than DNA (probe molecules are muchsmaller than template DNA molecules), the detection volume can beslightly smaller than the full reaction volume in order to detect eachand every copy of DNA initially present in the reactor at the start ofthe reaction. The detection volume may thus be 80% of, or as low as 50%of, the reaction volume.

The microfluidic PCR reactor used herein is typically a straightmicrochannel, 0.3 mm deep and 1.5 mm wide. Depending on the requiredreaction volume, the length of the reactor can be from 10 mm to 20 mm.The width of the channel can be varied from 100 microns to 3 mm to beable to use the same geometry of PCR heaters to maintain desiredtemperature uniformity and speed of heating (which depends on effectivethermal mass heated by the heaters). The depth of the channel can alsobe increased to 350 microns or 400 microns without incurring loss ofuniformity of temperature.

FIG. 1 shows a schematic cross-sectional view of a part of an apparatusas described herein, showing input of sample into a microfluidiccartridge 200 via a pipette 10 (such as a disposable pipette) and aninlet 202 in the cartridge. Suitable cartridges 200 are furtherdescribed herein but it is to be understood that the detection systemcan also be configured to detect analytes in microfluidic channels foundin situ in the apparatus. Such channels may therefore be fixed in theapparatus and reusable, for example by flushing through after each use,instead of being associated with a removable and disposable item such asa cartridge. Inlet 202 is preferably configured to receive a pipette orthe bottom end of a PCR tube and thereby accept sample for analysis withminimum waste, and with minimum introduction of air. Although notapparent from FIG. 1, several pipettes 10 may operate in parallel withone another to introduce multiple samples into cartridge 200. Cartridge200 is disposed on top of and in contact with a heater substrate 400. Adetector, as further described herein, comprises a read head 300 and acover 310. Read head 300 is positioned above cartridge 200, and a coverfor optics 310 restricts the amount of ambient light that can bedetected by the read head.

Fluorescence Detection System, Including Lenses and Filters, andMultiple Parallel Detection for a Multi-Lane Cartridge

The detection system herein is configured to monitor fluorescence comingfrom one or more species involved in a biochemical reaction. The systemcan be, for example, an optical detector having a light source thatselectively emits light in an absorption band of a fluorescent dye, anda light detector that selectively detects light in an emission band ofthe fluorescent dye, wherein the fluorescent dye corresponds to afluorescent polynucleotide probe or a fragment thereof, as furtherdescribed elsewhere herein. Alternatively, the optical detector caninclude a bandpass-filtered diode that selectively emits light in theabsorption band of the fluorescent dye and a bandpass filteredphotodiode that selectively detects light in the emission band of thefluorescent dye. For example, the optical detector can be configured toindependently detect a plurality of fluorescent dyes having differentfluorescent emission spectra, wherein each fluorescent dye correspondsto a fluorescent polynucleotide probe or a fragment thereof. Forexample, the optical detector can be configured to independently detecta plurality of fluorescent dyes at a plurality of different locationsof, for example, a microfluidic substrate, wherein each fluorescent dyecorresponds to a fluorescent polynucleotide probe or a fragment thereof.The detector further has potential for 2, 3 or 4 color detection and iscontrolled by software, preferably custom software, configured to sampleinformation from the detector.

The detection system described herein is capable of detecting afluorescence signal from nanoliter scale PCR reactions. Advantageously,the detector is formed from inexpensive components, having no movingparts. The detector can be configured to couple to a microfluidiccartridge as further described herein, and can also be part of apressure application system, such as a sliding lid on an apparatus inwhich the detector is situated, that keeps the cartridge in place.

FIGS. 2-4B depict an embodiment of a highly sensitive fluorescencedetection system that includes light emitting diodes (LED's),photodiodes, and filters/lenses for monitoring, in real-time, one ormore fluorescent signals emanating from the microfluidic channel. Theembodiment in FIGS. 2-4B displays a two-color detection system having amodular design that couples with a single microfluidic channel found,for example, in a microfluidic cartridge. It would be understood by oneskilled in the art that the description herein could also be adapted tocreate a detector that just detects a single color of light. FIGS. 4Aand 4B show elements of optical detector elements 1220 including lightsources 1232 (for example, light emitting diodes), lenses 1234, lightdetectors 1236 (for example, photodiodes) and filters 1238. The detectorcomprises two LED's (blue and red, respectively) and two photodiodes.The two LED's are configured to transmit a beam of focused light on to aparticular region of the cartridge. The two photodiodes are configuredto receive light that is emitted from the region of the cartridge. Onephotodiode is configured to detect emitted red light, and the otherphotodiode is configured to detect emitted blue light. Thus, in thisembodiment, two colors can be detected simultaneously from a singlelocation. Such a detection system can be configured to receive lightfrom multiple microfluidic channels by being mounted on an assembly thatpermits it to slide over and across the multiple channels. The filterscan be, for example, bandpass filters, the filters at the light sourcescorresponding to the absorption band of one or more fluorogenic probesand the filters at the detectors corresponding to the emission band ofthe fluorogenic probes.

FIGS. 5 and 6 show an exemplary read-head comprising a multiplexed 2color detection system that is configured to mate with a multi-lanemicrofluidic cartridge. FIG. 5 shows a view of the exterior of amultiplexed read-head. FIG. 6 is an exploded view that shows how variousdetectors are configured within an exemplary multiplexed read head, andin communication with an electronic circuit board.

Each of the detection systems multiplexed in the assembly of FIGS. 5 and6 is similar in construction to the embodiment of FIGS. 2-4B. The modulein FIGS. 5 and 6 is configured to detect fluorescence from each of 12microfluidic channels, as found in, for example, the respective lanes ofa 12-lane microfluidic cartridge. Such a module therefore comprises 24independently controllable detectors, arranged as 12 pairs of identicaldetection elements. Each pair of elements is then capable of dual-colordetection of a predetermined set of fluorescent probes. It would beunderstood by one of ordinary skill in the art that other numbers ofpairs of detectors are consistent with the apparatus described herein.For example, 4, 6, 8, 10, 16, 20, 24, 25, 30, 32, 36, 40, and 48 pairsare also consistent and can be configured according to methods andcriteria understood by one of ordinary skill in the art.

Detection Sensitivity, Time Constant and Gain

A typical circuit that includes a detector as described herein includes,in series, a preamplifier, a buffer/inverter, a filter, and a digitizer.Sensitivity is important so that high gain is very desirable. In oneembodiment of the preamplifier, a very large, for example 100 GΩ,resistor is placed in parallel with the diode. Other values of aresistor would be consistent with the technology herein: such valuestypically fall in the range 0.5-100 GΩ, such as 1-50 GΩ, or 2-10 GΩ. Anexemplary pre-amplifier circuit configured in this way is shown in FIG.7. Symbols in the figure have their standard meanings in electroniccircuit diagrams.

The FIG. 7 shows a current-to-voltage converter/pre-amplifier circuitsuitable for use with the detection system. D11 is the photodetectorthat collects the fluorescent light coming from the microfluidic channeland converts it into an electric current. The accompanying circuitry isused to convert these fluorescent currents into voltages suitable formeasurement and output as a final measure of the fluorescence.

A resistor-capacitor circuit in FIG. 7 contains capacitor C45 andresistor R25. Together, the values of capacitance of C45 and resistanceof R25 are chosen so as to impact the time constant τ_(c) (equal to theproduct of R25 and C45) of the circuit as well as gain of the detectioncircuit. The higher the time constant, the more sluggish is the responseof the system to incident light. It typically takes the duration of afew time constants for the photodetector to completely charge to itsmaximum current or to discharge to zero from its saturation value. It isimportant for the photo current to decay to zero between measurements,however. As the PCR systems described herein are intended to affordrapid detection measurements, the product R₂₅C₄₅ should therefore bemade as low as possible. However, the gain of the pre-amplifier whosecircuitry is shown is directly proportional to the(fluorescent-activated) current generated in the photodetector and theresistance R₂₅. As the fluorescence signal from the microfluidic channeldevice is very faint (due to low liquid volume as well as small pathlengths of excitation), it is thus important to maximize the value ofR₂₅. In some embodiments, R₂₅ is as high as 100 Giga-Ohms (for example,in the range 10-100 GΩ), effectively behaving as an open-circuit. Withsuch values, the time-constant can take on a value of approximately50-100 ms by using a low-value capacitor for C45. For example, thelowest possible available standard off-the-shelf capacitor has a valueof 1 pF (1 picoFarad). A time-constant in the range 50-100 ms ensuresthat the photocurrent decays to zero in approximately 0.5 s (approx. 6cycles) and therefore that approximately 2 samplings can be made persecond. Other time constants are consistent with effective use of thetechnology herein, such as in the range 10 ms-1 s, or in the range 50ms-500 ms, or in the range 100-200 ms. The actual time constant suitablefor a given application will vary according to circumstance and choiceof capacitor and resistor values. Additionally, the gain achieved by thepre-amplifier circuit herein may be in the range of 10⁷-5×10⁹, forexample may be 1×10⁹.

As the resistance value for R25 is very high (˜100 GΩ), the manner ofassembly of this resistor on the optics board is important for theoverall efficiency of the circuit. Effective cleaning of the circuitduring assembly and before use is important to achieve an optimaltime-constant and gain for the optics circuit.

It is also important to test each photo-diode that is used, because manydo not perform according to specification.

Sensitivity and Aperturing

The LED light passes through a filter before passing through the samplein the micro-fluidic channel (as described herein, typically 300 gdeep). This is a very small optical path-length for the light in thesample. The generated fluorescence then also goes through a secondfilter, and into a photo-detector. Ultimately, then, the detector mustbe capable of detecting very little fluorescence. Various aspects of thedetector configuration can improve sensitivity, however.

The illumination optics can be designed so that the excitation lightfalling on the PCR reactor is incident along an area that is similar tothe shape of the reactor. As the reactor is typically long and narrow,the illumination spot should be long and narrow, i.e., extended, aswell. The length of the spot can be adjusted by altering a number offactors, including: the diameter of the bore where the LED is placed(the tube that holds the filter and lens has an aperturing effect); thedistance of the LED from the PCR reactor; and the use of proper lens atthe right distance in between. As the width of the beam incident on thereactor is determined by the bore of the optical element (approximately6 mm in diameter), it is typical to use an aperture (a slit having awidth approximately equal to the width of the reactor, and a lengthequal to the length of the detection volume) to make an optimalillumination spot. A typical spot, then, is commensurate with thedimensions of a PCR reaction chamber, for example 1.5 mm wide by 7 mmlong. FIG. 8A shows the illumination spot across 12 PCR reactors for thetwo different colors used. A typical aperture is made of anodizedaluminum and has very low autofluoresence in the wavelengths ofinterest. FIG. 8B illustrates a cross-section of a detector, showing anexemplary location for an aperture 802.

The optimal spot size and intensity is importantly dependent on theability to maintain the correct position of the LED's with respect tothe center of the optical axis. Special alignment procedures and checkscan be utilized to optimize this. The different illuminations can alsobe normalized with respect to each other by adjusting the power currentthrough each of the LED's or adjusting the fluorescence collection time(the duration for which the photodetector is on before measuring thevoltage) for each detection spot. It is also important to align thedetectors with the axis of the micro-channels.

The aperturing is also important for successful fluorescence detectionbecause as the cross-sectional area of the incident beam increases insize, so the background fluorescence increases, and the fluorescenceattributable only to the molecules of interest (PCR probes) gets masked.Thus, as the beam area increases, the use of an aperture increases theproportion of collected fluorescence that originates only from the PCRreactor. Note that the aperture used in the detector herein not onlyhelps collect fluorescence only from the reaction volume but itcorrespondingly adjusts the excitation light to mostly excite thereaction volume. The excitation and emission aperture is, of course, thesame.

Based on a typical geometry of the optical exctiation and emissionsystem and aperturing, show spot sizes as small as 0.5 mm by 0.5 mm andas long as 8 mm×1.5 mm have been found to be effective. By using a longdetector (having an active area 6 mm by 1 mm) and proper lensing, theaperture design can extend the detection spot to as long as 15-20 mm,while maintaining a width of 1-2 mm using an aperture. Correspondingly,the background fluorescence decreases as the spot size is decreased,thereby increasing the detection sensitivity.

Use of Detection System to Measure/Detect Fluid in PCR Chamber

The fluorescence detector is sensitive enough to be able to collectfluorescence light from a PCR chamber of a microfluidic substrate. Thedetector can also be used to detect the presence of liquid in thechamber, a measurement that provides a determination of whether or notto carry out a PCR cycle for that chamber. For example, in amulti-sample cartridge, not all chambers will have been loaded withsample; for those that are not, it would be unnecessary to apply aheating protocol thereto. One way to determine presence or absence of aliquid is as follows. A background reading is taken prior to filling thechamber with liquid. Another reading is taken after microfluidicoperations have been performed that should result in filling the PCRchamber with liquid. The presence of liquid alters the fluorescencereading from the chamber. A programmable threshold value can be used totune an algorithm programmed into a processor that controls operation ofthe apparatus as further described herein (for example, the secondreading has to exceed the first reading by 20%). If the two readings donot differ beyond the programmed margin, the liquid is deemed to nothave entered the chamber, and a PCR cycle is not initiated for thatchamber. Instead, a warning is issued to a user.

Microfluidic Cartridge

Where the microfluidic channels that contain analytes detected by thedetection system are situated in a microfluidic cartridge, the cartridgetypically has attributes as follows. The microfluidic cartridge isdesigned so that it receives thermal energy from one or more heatingelements present in the heater unit described elsewhere herein when itis in thermal communication therewith. The heater unit may be part of anapparatus, configured to receive the cartridge, and comprising otherfeatures such as control circuitry, user interface, and detector, aswell as still other features. An exemplary such apparatus is furtherdescribed herein; additional embodiments of such a apparatus are foundin U.S. patent application Ser. No. ______, entitled “MicrofluidicSystem for Amplifying and Testing Polynucleotides in Parallel”, andfiled on even date herewith, the specification of which is incorporatedherein by reference.

By cartridge is meant a unit that may be disposable, or reusable inwhole or in part, and that is configured to be used in conjunction withsome other apparatus that has been suitably and complementarilyconfigured to receive and operate on (such as deliver energy to via aheater module as described herein) the cartridge.

One aspect of the present technology relates to a detector that isconfigured to selectively detect analytes in a microfluidic cartridgehaving two or more sample lanes arranged so that analyses can be carriedout in two or more of the lanes in parallel, for example simultaneously,and wherein each lane is independently associated with a given sample.

A sample lane, as found in a microfluidic cartridge, is an independentlycontrollable set of elements by which a sample can be analyzed, forexample by carrying out PCR on a sample in which the presence or absenceof one or more polynucleotides is to be determined, according to methodsdescribed in, e.g., U.S. patent application Ser. No. ______, entitled“Microfluidic Cartridge and Method of Making Same”, and filed on evendate herewith. A sample lane comprises at least a sample inlet, and amicrofluidic network having one or more microfluidic components, asfurther described herein.

In various embodiments, a sample lane of a microfluidic cartridge caninclude a sample inlet port or valve, and a microfluidic network thatcomprises, in fluidic communication one or more components selected fromthe group consisting of: at least one thermally actuated valve, a bubbleremoval vent, at least one gate, at least one thermally actuated pump, adownstream thermally actuated valve, mixing channels, one or morepositioning elements, and a PCR reaction zone. The detector describedherein can be configured to detect light emitted from any one of theforegoing microfluidic components, but is typically configured to detectlight from a reaction chamber.

In various embodiments, the microfluidic network can be configured tocouple heat from an external heat source, such as provided by a heaterunit described elsewhere herein, to a sample mixture comprising PCRreagent and neutralized polynucleotide sample under thermal cyclingconditions suitable for creating PCR amplicons from the neutralizedpolynucleotide sample.

A multi-lane cartridge is typically configured to accept a number ofsamples in series or in parallel, in particular embodiments 12 samplesand in other embodiments 24, or 48 samples, wherein the samples includeat least a first sample and a second sample, wherein the first sampleand the second sample each contain one or more polynucleotides in a formsuitable for amplification. The polynucleotides in question may be thesame as, or different from one another, in different samples and hencein different lanes of the cartridge. The cartridge typically processeseach sample by increasing the concentration of a polynucleotide to bedetermined and/or by reducing the concentration of inhibitors relativeto the concentration of polynucleotide to be determined.

Exemplary Microfluidic Cartridges

FIG. 9A shows a perspective view of a portion of an exemplarymicrofluidic cartridge 200 according to the present technology. FIG. 9Bshows a close-up view of a portion of the cartridge 200 of FIG. 9Aillustrating various representative components. The cartridge 200 may bereferred to as a multi-lane PCR cartridge with dedicated sample inlets202. For example sample inlet 202 is configured to accept a liquidtransfer member (not shown) such as a syringe, a pipette, or a PCR tubecontaining a PCR ready sample. More than one inlet 202 is shown in FIGS.9A, 9B, wherein one inlet operates in conjunction with a single samplelane. Various components of microfluidic circuitry in each lane are alsovisible. For example, microvalves 204, and 206, and vents 208, are partsof microfluidic circuitry in a given lane. Microfluidic circuitry istypically disposed within a microfluidic substrate found in one or morelayers of the cartridge. Also shown is an ultrafast PCR reactor 210,which, as further described herein, is a microfluidic channel in a givensample lane that is long enough to permit PCR to amplify polynucleotidespresent in a sample. Above each PCR reactor 210 is a window 212 thatpermits detection of fluorescence from a fluorescent substance in PCRreactor 210 when a detector is situated above window 212. It is to beunderstood that other configurations of windows are possible including,but not limited to, a single window that straddles each PCR reactoracross the width of cartridge 200.

A multi-sample cartridge comprises at least a first microfluidic networkand a second microfluidic network, adjacent to one another, wherein eachof the first microfluidic network and the second microfluidic network isas elsewhere described herein, and wherein the first microfluidicnetwork accepts the first sample, and wherein the second microfluidicnetwork accepts the second sample.

In some embodiments, the multi-sample cartridge has a size substantiallythe same as that of a 96-well plate as is customarily used in the art.Advantageously, then, the cartridge may be used with plate handlers usedelsewhere in the art.

The sample inlets of adjacent sample lanes in a multi-sample cartridgeare reasonably spaced apart from one another to prevent anycontamination of one sample inlet from another sample when a userintroduces a sample into any one cartridge. In an embodiment, the sampleinlets are configured so as to prevent subsequent inadvertentintroduction of sample into a given lane after a sample has already beenintroduced into that lane. Thus, the elements of the detector describedherein are engineered to be compatible with the overall cartridge sizeand the separation between the respective lanes.

In other embodiments, the multi-sample cartridge is designed so that aspacing between the centroids of sample inlets is 9 mm, which is anindustry-recognized standard. This means that, in certain embodimentsthe center-to-center distance between inlet holes in the cartridge thataccept samples from PCR tubes, as further described herein, is 9 mm. Theinlet holes are manufactured conical in shape with an appropriateconical angle so that industry-standard pipette tips (2 μl, 20 μl, 200μl, volumes, etc.) fit snugly. The apparatus herein may be adapted tosuit other, later-arising, industry standards not otherwise describedherein.

FIG. 10 shows a plan view of an exemplary microfluidic cartridge having12 lanes. The inlet ports have a 6 mm spacing, so that, when used inconjunction with an automated sample loader having 4 heads, spacedequidistantly at 18 mm apart, the inlets can be loaded in three batchesof 4 inlets: e.g., inlets 1, 4, 7, and 10 together, followed by 2, 5, 8,and 1, then finally 3, 6, 9, and 12, wherein the 12 inlets are numberedconsecutively from one side of the cartridge to the other.

In use, cartridge 200 is typically thermally associated with an array ofheat sources configured to operate the components (e.g., valves, gates,actuators, and processing region 210) of the device. Particularcomponents of exemplary microfluidic networks are further described inU.S. patent application Ser. No. ______, entitled “MicrofluidicCartridge and Method of Making Same” and filed on even date herewith.

FIG. 11 shows a plan view of a representative microfluidic circuit foundin one lane of a multi-lane cartridge such as shown in FIG. 10. Otherconfigurations of microfluidic network would be consistent with thefunction of the cartridges and apparatus described herein. In sequence,sample is introduced through liquid inlet 202, flows into a bubbleremoval vent channel 208 (which permits adventitious air bubblesintroduced into the sample during entry, to escape), and continues alonga channel 216. Throughout the operation of cartridge 200 the fluid ismanipulated as a microdroplet (not shown in FIG. 5), and the variousmicrofluidic components are actuated or controlled by application ofheat from the heater unit further described herein. Valves 204 and 206are initially open, so that a microdroplet of sample-containing fluidcan be pumped into PCR reactor channel 210 from inlet hole 202. Uponinitiating of processing, the detector present on top of the PCR reactorchecks for the presence of liquid in the PCR channel, and then closesvalves 204 and 206 to isolate the PCR reaction mix from the outside. Thereactor 210 is a microfluidic channel that is heated through a series ofcycles to carry out amplification of nucleotides in the sample, asfurther described herein. Both valves 204 and 206 are closed prior tothermocycling to prevent any evaporation of liquid, bubble generation,or movement of fluid from the PCR reactor. End vent 214 prevents a userfrom introducing any excess amount of liquid into the microfluidiccartridge, as well as playing a role of containing any sample fromspilling over to unintended parts of the cartridge. A user may inputsample volumes as small as an amount to fill from the bubble removalvent to the middle of the microreactor, or up to valve 204 or beyondvalve 204. The use of microvalves prevents both loss of liquid or vaporthereby enabling even a partially filled reactor to successfullycomplete a PCR thermocycling reaction.

The cartridge can further include a heat sealable laminate layer(typically between about 100 and about 125 microns thick) attached tothe bottom surface of a microfluidic substrate using, for example, heatbonding. The cartridge can further include a thermal interface materiallayer (typically about 125 microns thick), attached to the bottom of theheat sealable laminate layer using, for example, pressure sensitiveadhesive. This layer can be compressible and have a higher thermalconductivity than common plastics, thereby serving to transfer heatacross the membrane more efficiently to the components of themicrofluidic network.

The application of pressure to contact the cartridge to the heater unitassists in achieving better thermal contact between the heater and theheat-receivable parts of the cartridge, and also prevents the bottomlaminate structure—where present—from expanding, as would happen if thePCR channel was partially filled with liquid so that the entrapped airwould be thermally expanded during thermocycling.

Further aspects of a microfluidic cartridge that adapt it to carryingout PCR efficiently are described in U.S. patent application Ser. No.______, entitled “Microfluidic Cartridge and Method of Making Same” andfiled on even date herewith, the specification of which is incorporatedherein by reference.

Microfluidic cartridge 200 can be fabricated as desired, for example,according to methods described in U.S. patent application Ser. No.______, entitled “Microfluidic Cartridge and Method of Making Same” andfiled on even date herewith.

Highly Multiplexed Cartridge Embodiments

Embodiments of the apparatus and cartridge described herein may beconstructed that have high-density microfluidic circuitry on a singlecartridge that thereby permit processing of multiple samples inparallel, or in sequence, on a single cartridge. Preferred numbers ofsuch multiple samples include 24, 36, 40, 48, 50, 60, 64, 72, 80, 84,96, and 100, but it would be understood that still other numbers areconsistent with the apparatus and cartridge herein, where deemedconvenient and practical.

Accordingly, different configurations of lanes, sample inlets, andassociated heater networks are contemplated that can facilitateprocessing such numbers of samples on a single cartridge are within thescope of the instant disclosure. Similarly, alternative configurationsof detectors and heating elements for use in conjunction with such ahighly multiplexed cartridge are also within the scope of thedescription herein.

It is also to be understood that the microfluidic cartridges andsubstrates described herein are not to be limited to rectangularconfigurations, but can include cartridges having circular, elliptical,triangular, rhombohedral, square, and other shapes. Such shapes may alsobe adapted to include some irregularity, such as a cut-out, tofacilitate placement in a complementary apparatus as further describedelsewhere herein.

FIG. 12 shows a representative 48-sample cartridge, and having an inletconfiguration different from others described and depicted herein. Theinlet configuration is exemplary and has been designed to maximizeefficiency of space usage on the cartridge. The inlet configuration canbe compatible with an automatic pipetting machine that has dispensingheads situated at a 9 mm spacing. For example, such a machine having 4heads can load 4 inlets at once, in 12 discrete steps, for the cartridgeof FIG. 12. Other configurations of inlets though not explicitlydescribed are compatible with the technology described herein.

In an exemplary embodiment, a highly multiplexed cartridge has 48 samplelanes, and permits independent control of each valve in each lane, with2 banks of thermocycling protocols per lane, as shown in FIG. 13. Thispermits samples to be loaded into the cartridge at different times, andpassed to the PCR reaction chambers independently of one another. Suchembodiments permit batch processing of PCR samples, where multiplesamples from different lanes are amplified by the same set ofheating/cooling cycles. For example, the PCR heaters can be arranged in2 banks (the heater arrays on the left and right are not in electricalcommunication with one another), thereby permitting a separate degree ofsample control.

FIG. 14 shows an embodiment of a radially-configured highly-multiplexedcartridge, having a number of inlets, microfluidic lanes, and PCRreaction chambers.

The various embodiments shown in FIGS. 12-14 are compatible with liquiddispensers, receiving bays, heater units, and detectors that areconfigured differently from the other specific examples describedherein. For example, such detectors may be configured to detect lightfrom multiple sample lanes simultaneously, or to scan over sample lanessingly or in batches successively.

In another preferred embodiment (not shown in the FIGs.), a cartridgeand apparatus is configured so that the read-head does not cover thesample inlets, thereby permitting loading of separate samples whileother samples are undergoing PCR thermocycling.

PCR Reagent Mixtures

PCR reagent mixes, and methods of preparation and use thereof, aregenerally known in the art. Herein, general aspects of such methods aredescribed, as they can be used with the apparatus and detection system.In various embodiments, the sample for introduction into a lane of thecartridge can include a PCR reagent mixture comprising a polymeraseenzyme and a plurality of nucleotides, and at least one probe thatselectively emits light detected by the detection system herein.

In various embodiments, preparation of a PCR-ready sample for use withan apparatus and cartridge as described herein can include contacting aneutralized polynucleotide sample with a PCR reagent mixture comprisinga polymerase enzyme and a plurality of nucleotides (in some embodiments,the PCR reagent mixture can further include a positive control plasmidand a fluorogenic hybridization probe selective for at least a portionof the plasmid). Other aspects of suitable PCR reagent mixture, forexample lyophilized formulations, are described in U.S. patentapplication Ser. No. ______, entitled “Microfluidic Cartridge and Methodof Making Same”, filed on even date herewith.

In various embodiments, the PCR-ready sample can include at least oneprobe that is selective for a polynucleotide sequence, e.g., thepolynucleotide sequence that is characteristic of a pathogen selectedfrom the group consisting of gram positive bacteria, gram negativebacteria, yeast, fungi, protozoa, and viruses. Steps by which such aPCR-ready sample is prepared involve contacting the neutralizedpolynucleotide sample or a PCR amplicon thereof with the probe. Theprobe can be a fluorogenic hybridization probe. The fluorogenichybridization probe can include a polynucleotide sequence coupled to afluorescent reporter dye and a fluorescence quencher dye.

In various embodiments, the PCR-ready sample further includes a samplebuffer.

In various embodiments, the PCR reagent mixture can further include apolymerase enzyme, a positive control plasmid and a fluorogenichybridization probe selective for at least a portion of the plasmid.

It is envisaged that the detection system herein is operable with anyprobe suitable for use in detecting a particular polynucleotide. Thechoice and use of a suitable probe is within the capability of oneskilled in the art. In various embodiments, the probe can be selectivefor a polynucleotide sequence that is characteristic of an organism, forexample any organism that employs deoxyribonucleic acid or ribonucleicacid polynucleotides. Thus, the probe can be selective for any organism.Suitable organisms include mammals (including humans), birds, reptiles,amphibians, fish, domesticated animals, wild animals, extinct organisms,bacteria, fungi, viruses, plants, and the like. The probe can also beselective for components of organisms that employ their ownpolynucleotides, for example mitochondria. In some embodiments, theprobe is selective for microorganisms, for example, organisms used infood production (for example, yeasts employed in fermented products,molds or bacteria employed in cheeses, and the like) or pathogens (e.g.,of humans, domesticated or wild mammals, domesticated or wild birds, andthe like). In some embodiments, the probe is selective for organismsselected from the group consisting of gram positive bacteria, gramnegative bacteria, yeast, fungi, protozoa, and viruses.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organism selected from the groupconsisting of Staphylococcus spp., e.g., S. epidermidis, S. aureus,Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistantStaphylococcus; Streptococcus(e.g., α, β or γ-hemolytic, Group A, B, C,D or G) such as S. pyogenes, S. agalactiae; E. faecalis, E. durans, andE. faecium (formerly S. faecalis, S. durans, S. faecium);nonenterococcal group D streptococci, e.g., S. bovis and S. equines;Streptococci viridans, e.g., S. mutans, S. sanguis, S. salivarius, S.mitior, A. milleri, S. constellatus, S. intermedius, and S. anginosus;S. iniae; S. pneumoniae; Neisseria, e.g., N. meningitides, N.gonorrhoeae, saprophytic Neisseria sp; Erysipelothrix, e.g., E.rhusiopathiae; Listeria spp., e.g., L. monocytogenes, rarely L. ivanoviiand L. seeligeri; Bacillus, e.g., B. anthracis, B. cereus, B. subtilis,B. subtilus niger, B. thuringiensis; Nocardia asteroids; Legionella,e.g., L. pneumonophilia, Pneumocystis, e.g., P. carinii;Enterobacteriaceae such as Salmonella, Shigella, Escherichia (e.g., E.coli, E. coliO157:H7); Klebsiella, Enterobacter, Serratia, Proteus,Morganella, Providencia, Yersinia, and the like, e.g., Salmonella, e.g.,S. typhi S. paratyphi A, B (S. schottmuelleri), and C (S. hirschfeldii),S. dublin S. choleraesuis, S. enteritidis, S. typhimurium, S.heidelberg, S. newport, S. infantis, S. agona, S. montevideo, and S.saint-paul; Shigella e.g., subgroups: A, B, C, and D, such as S.flexneri, S. sonnei, S. boydii, S. dysenteriae; Proteus (P. mirabilis,P. vulgaris, and P. myxofaciens), Morganella (M. morganii); Providencia(P. rettgeri, P. alcalifaciens, and P. stuartii); Yersinia, e.g., Y.pestis, Y. enterocolitica; Haemophilus, e.g., H. influenzae, H.parainfluenzae H. aphrophilus, H. ducreyi; Brucella, e.g., B. abortus,B. melitensis, B. suis, B. canis; Francisella, e.g., F. tularensis;Pseudomonas, e.g., P. aeruginosa, P. paucimobilis, P. putida, P.fluorescens, P. acidovorans, Burkholderia (Pseudomonas) pseudomallei,Burkholderia mallei, Burkholderia cepacia and Stenotrophomonasmaltophilia; Campylobacter, e.g., C. fetus fetus, C. jejuni, C. pylori(Helicobacter pylori); Vibrio, e.g., V. cholerae, V. parahaemolyticus,V. mimicus, V. alginolyticus, V. hollisae, V. vulnificus, and thenonagglutinable vibrios; Clostridia, e.g., C. perfingens, C. tetani, C.difficile, C. botulinum; Actinomyces, e.g., A. israelii; Bacteroides,e.g., B. fragilis, B. thetaiotaomicron, B. distasonis, B. vulgatus, B.ovatus, B. caccae, and B. merdae; Prevotella, e.g., P. melaminogenica;genus Fusobacterium; Treponema, e.g. T. pallidum subspecies endemicum,T. pallidum subspecies pertenue, T. carateum, and T. pallidum subspeciespallidum; genus Borrelia, e.g., B burgdorferi; genus Leptospira;Streptobacillus, e.g., S. moniliformis; Spirillum, e.g., S. minus;Mycobacterium, e.g., M. tuberculosis, M. bovis, M. africanum, M. aviumM. intracellulare, M. kansasii, M. xenopi, M. marinum, M. ulcerans, theM. fortuitum complex (M. fortuitum and M. chelonei), M. leprae, M.asiaticum, M. chelonei subsp. abscessus, M. fallax, M. fortuitum, M.malmoense, M. shimoidei, M. simiae, M. szulgai, M. xenopi; Mycoplasma,e.g., M. hominis, M. orale, M. salivarium, M. fermentans, M. pneumoniae,M. bovis, M. tuberculosis, M. avium, M. leprae; Mycoplasma, e.g., M.genitalium; Ureaplasma, e.g., U. urealyticum; Trichomonas, e.g., T.vaginalis; Cryptococcus, e.g., C. neoformans; Histoplasma, e.g., H.capsulatum; Candida, e.g., C. albicans; Aspergillus sp; Coccidioides,e.g., C. immitis; Blastomyces, e.g. B. dermatitidis; Paracoccidioides,e.g., P. brasiliensis; Penicillium, e.g., P. marneffei; Sporothrix,e.g., S. schenckii; Rhizopus, Rhizomucor, Absidia, and Basidiobolus;diseases caused by Bipolaris, Cladophialophora, Cladosporium,Drechslera, Exophiala, Fonsecaea, Phialophora, Xylohypha, Ochroconis,Rhinocladiella, Scolecobasidium, and Wangiella; Trichosporon, e.g., T.beigelii; Blastoschizomyces, e.g., B. capitatus; Plasmodium, e.g., P.falciparum, P. vivax, P. ovale, and P. malariae; Babesia sp; protozoa ofthe genus Trypanosoma, e.g., T. cruzi; Leishmania, e.g., L. donovani, L.major L. tropica, L. mexicana, L. braziliensis, L. viannia braziliensis;Toxoplasma, e.g., T. gondii; Amoebas of the genera Naegleria orAcanthamoeba; Entamoeba histolytica; Giardia lamblia; genusCryptosporidium, e.g., C. parvum; Isospora belli; Cyclosporacayetanensis; Ascaris lumbricoides; Trichuris trichiura; Ancylostomaduodenale or Necator americanus; Strongyloides stercoralis Toxocara,e.g., T. canis, T. cati; Baylisascaris, e.g., B. procyonis; Trichinella,e.g., T. spiralis; Dracunculus, e.g., D. medinensis; genus Filarioidea;Wuchereria bancrofti; Brugia, e.g., B. malayi, or B. timori; Onchocercavolvulus; Loa loa; Dirofilaria immitis; genus Schistosoma, e.g., S.japonicum, S. mansoni, S. mekongi, S. intercalatum, S. haematobium;Paragonimus, e.g., P. Westermani, P. Skriabini; Clonorchis sinensis;Fasciola hepatica; Opisthorchis sp; Fasciolopsis buski; Diphyllobothriumlatum; Taenia, e.g., T. saginata, T. solium; Echinococcus, e.g., E.granulosus, E. multilocularis; Picornaviruses, rhinoviruses echoviruses,coxsackieviruses, influenza virus; paramyxoviruses, e.g., types 1, 2, 3,and 4; adnoviruses; Herpesviruses, e.g., HSV-1 and HSV-2;varicella-zoster virus; human T-lymphotrophic virus (type I and typeII); Arboviruses and Arenaviruses; Togaviridae, Flaviviridae,Bunyaviridae, Reoviridae; Flavivirus; Hantavirus; Viral encephalitis(alphaviruses [e.g., Venezuelan equine encephalitis, eastern equineencephalitis, western equine encephalitis]); Viral hemorrhagic fevers(filoviruses [e.g., Ebola, Marburg] and arenaviruses [e.g., Lassa,Machupo]); Smallpox (variola); retroviruses e.g., human immunodeficiencyviruses 1 and 2; human papillomavirus [HPV] types 6, 11, 16, 18, 31, 33,and 35.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organisms selected from the groupconsisting of Pseudomonas aeruginosa, Proteus mirabilis, Klebsiellaoxytoca, Klebsiella pneumoniae, Escherichia coli, AcinetobacterBaumannii, Serratia marcescens, Enterobacter aerogenes, Enterococcusfaecium, vancomycin-resistant enterococcus (VRE), Staphylococcus aureus,methecillin-resistant Staphylococcus aureus (MRSA), Streptococcusviridans, Listeria monocytogenes, Enterococcus spp., Streptococcus GroupB, Streptococcus Group C, Streptococcus Group G, Streptococcus Group F,Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcusepidermidis, Gardenerella vaginalis, Micrococcus sps., Haemophilusinfluenzae, Neisseria gonorrhoeee, Moraxella catarrahlis, Salmonellasps., Chlamydia trachomatis, Peptostreptococcus productus,Peptostreptococcus anaerobius, Lactobacillusfermentum, Eubacteriumlentum, Candida glabrata, Candida albicans, Chlamydia spp., Camplobacterspp., Salmonella spp., smallpox (variola major), Yersina Pestis, HerpesSimplex Virus I (HSV I), and Herpes Simplex Virus II (HSV II).

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of Group B Streptococcus.

In various embodiments, a method of carrying out PCR on a sample canfurther include one or more of the following steps: heating thebiological sample in a microfluidic channel; pressurizing the biologicalsample in the microfluidic channel at a pressure differential comparedto ambient pressure of between about 20 kilopascals and 200 kilopascals,or in some embodiments between about 70 kilopascals and 110 kilopascals.

In some embodiments, the method for sampling a polynucleotide caninclude the steps of: placing a microfluidic cartridge containing aPCR-ready sample in a receiving bay of a suitably configured apparatus;carrying out PCR on the sample under thermal cycling conditions suitablefor creating PCR amplicons from the neutralized polynucleotide in thesample, the PCR-ready sample comprising a polymerase enzyme, a positivecontrol plasmid, a fluorogenic hybridization probe selective for atleast a portion of the plasmid, and a plurality of nucleotides;contacting the neutralized polynucleotide sample or a PCR ampliconthereof with the at least one fluorogenic probe that is selective for apolynucleotide sequence, wherein the probe is selective for apolynucleotide sequence that is characteristic of an organism selectedfrom the group consisting of gram positive bacteria, gram negativebacteria, yeast, fungi, protozoa, and viruses; and detecting thefluorogenic probe, the presence of the organism for which the onefluorogenic probe is selective is determined.

Carrying out PCR on a PCR-ready sample can additionally include:independently contacting each of the neutralized polynucleotide sampleand a negative control polynucleotide with the PCR reagent mixture underthermal cycling conditions suitable for independently creating PCRamplicons of the neutralized polynucleotide sample and PCR amplicons ofthe negative control polynucleotide; and/or contacting the neutralizedpolynucleotide sample or a PCR amplicon thereof and the negative controlpolynucleotide or a PCR amplicon thereof with at least one probe that isselective for a polynucleotide sequence.

In various embodiments, a method of using the apparatus and detectionsystem described herein can further include one or more of the followingsteps: determining the presence of a polynucleotide sequence in thebiological sample, the polynucleotide sequence corresponding to theprobe, if the probe is detected in the neutralized polynucleotide sampleor a PCR amplicon thereof; determining that the sample was contaminatedif the probe is detected in the negative control polynucleotide or a PCRamplicon thereof; and/or in some embodiments, wherein the PCR reagentmixture further comprises a positive control plasmid and a plasmid probeselective for at least a portion of the plasmid, the method furtherincluding determining that a PCR amplification has occurred if theplasmid probe is detected.

Heater Configurations to Ensure Uniform Heating of a Region

In general, the microfluidic channels in which the presence or absenceof one or more analytes is determined by the detection system describedherein are disposed in thermal contact with a dedicated heater unit. Forexample, the microfluidic cartridges described herein are typicallyconfigured to position in a complementary receiving bay in an apparatusthat contains a heater unit. The heater unit is configured to deliverheat to specific microfluidic channels, such as specific regions of thecartridge, including but not limited to one or more microfluidiccomponents, at specific times. For example, the heat source isconfigured so that particular heating elements are situated adjacent tospecific components of a microfluidic network. In certain embodiments,the apparatus uniformly controls the heating of a region of amicrofluidic network. In an exemplary embodiment, multiple heaters canbe configured to simultaneously and uniformly heat a region, such as thePCR reaction chamber, of a microfluidic network.

Generally, the heating of microfluidic components, such as a PCRreaction chamber, is controlled by passing currents through suitablyconfigured microfabricated heaters. Under control of suitable circuitry,the lanes of a multi-lane cartridge can then be controlled independentlyof one another. This can lead to a greater energy efficiency of theapparatus, because not all heaters are heating at the same time, and agiven heater is receiving current for only that fraction of the timewhen it is required to heat. Control systems and methods of controllablyheating various heating elements are further described in U.S. patentapplication Ser. No. ______, filed Nov. 14, 2007 and entitled “HeaterUnit for Microfluidic Diagnostic System”.

FIG. 15 shows a cross-sectional view of an exemplary microfluidiccartridge to show the location of a PCR channel in relation to theheaters when the cartridge is placed in a suitable apparatus. The viewin FIG. 15 is also referred to as a sectional-isometric view of thecartridge lying over a heater wafer. A window 903 above the PCR channelin the cartridge is shown in perspective view. PCR channel 901 (forexample, 150μ deep×700μ wide), is shown in an upper layer of thecartridge. A laminate layer 905 of the cartridge (for example, 125μthick) is directly under the PCR channel 901. A further layer of thermalinterface laminate 907 on the cartridge (for example, 125μ thick) liesdirectly under the laminate layer 905. Heaters 909, 911 are situated ina further substrate layer 913 directly under the thermal interfacelaminate, shown in cross-section. In one embodiment the heaters arephotolithographically defined and etched metal layers of gold (typicallyabout 3,000 Å thick).

An exemplary such heater array is shown in FIGS. 16A and 16B. Additionalembodiments of heater arrays are described in U.S. patent applicationSer. No. ______, entitled “Heater Unit for Microfluidic DiagnosticSystem” and filed on even date herewith, the specification of which isincorporated herein by reference in its entirety.

Referring to FIGS. 16A and 16B, the PCR reaction chamber 1001, typicallyhaving a volume ˜1.6 μl, is configured with a long side and a shortside, each with an associated heating element. The apparatus thereforeincludes four heaters disposed along the sides of, and configured toheat, the PCR reaction chamber, as shown in the exemplary embodiment ofFIG. 16A: long top heater 1005, long bottom heater 1003, short leftheater 1007, and short right heater 1009. The small gap between long topheater 1005 and long bottom heater 1003 results in a negligibletemperature gradient (a difference of less than 1° C. across the widthof the PCR channel at any point along the length of the PCR reactionchamber) and therefore an effectively uniform temperature throughout thePCR reaction chamber. The heaters on the short edges of the PCR reactorprovide heat to counteract the gradient created by the two long heatersfrom the center of the reactor to the edge of the reactor. It would beunderstood by one of ordinary skill in the art that still otherconfigurations of one or more heater(s) situated about a PCR reactionchamber are consistent with the methods and apparatus described herein.For example, a ‘long’ side of the reaction chamber can be configured tobe heated by two or more heaters. Specific orientations andconfigurations of heaters are used to create uniform zones of heatingeven on substrates having poor thermal conductivity because the poorthermal conductivity of glass, or quartz, or fused silica substrates isutilized to help in the independent operation of various microfluidiccomponents such as valves and independent operation of the various PCRlanes.

The configuration for uniform heating, shown in FIG. 16A for a singlePCR reaction chamber, can be applied to a multi-lane PCR cartridge inwhich multiple independent PCR reactions occur. See, e.g., FIG. 16C.

Alignment of microheaters in the heater module with correspondingheat-requiring microcomponents (such as valves, pumps, gates, reactionchambers, etc). The microheaters can be designed to be slightly biggerthan the heat requiring microfluidic components so that even though thecartridge may be off-centered from the heater, the individual componentscan still function effectively.

In other embodiments, as further described in U.S. patent applicationSer. No. ______, filed Nov. 14, 2007 and entitled “Heater Unit forMicrofluidic Diagnostic System”, the heaters may have an associatedtemperature sensor, or may themselves function as sensors,

Exemplary Electronics and Software

FIG. 17 describes exemplary electronics architecture modules. It wouldbe understood by one of ordinary skill in the art that otherconfigurations of electronics components are consistent with operationof the apparatus as described herein. In the exemplary embodiment, theelectronics architecture is distributed across two components of theapparatus: the Analyzer 2100 and a Heater Assembly 2102. The Analyzercontains an Optical Detection Unit 2108, a Control Board 2114, aBackplane 2112, and a LCD Touchscreen 2110. The Control Board includes aCard Engine 2116 further described herein, and Compact Flash memory2118, as well as other components. The Heater Assembly includes a HeaterBoard 2104 and a Heater Mux Board 2106, both further describedelsewhere, for example, in U.S. patent application Ser. No. ______,filed on even date herewith and entitled “Heater Unit for MicrofluidicDiagnostic System”.

In the exemplary embodiment, the Card Engine electronics module 2116 isa commercial, off the shelf “single board computer” containing aprocessor, memory, and flash memory for operating system storage.

The LCD+Touchscreen electronics module 2110 is a user interface, forexample, driven through a 640 pixel by 480 pixel 8 inch LCD and 5-wiretouchscreen.

The Compact Flash electronics module 2118 is a 256 megabyte commercial,off the shelf, compact flash module for application and data storage.Other media are alternatively usable, such as USB-drive, smart mediacard, memory stick, and smart data-card having the same or other storagecapacities.

The Backplane electronics module 2112 is a point of connection for theremovable heater assembly 2102. Bare PC boards with two connectors aresufficient to provide the necessary level of connectivity.

The Control Board electronics module 2114 supports peripherals to theCard Engine electronics module 2116. In the exemplary embodiment, theperipherals include such devices as a USB host+slave or hub, a USB CDROMinterface, serial ports, and ethernet ports. The Control Board 2114 caninclude a power monitor with a dedicated processor. The Control Boardmay also include a real time clock. The Control Board may furtherinclude a speaker. The Control Board 2114 also includes a CPLD toprovide SPI access to all other modules and programming access to allother modules. The Control Board includes a programmable high voltagepower supply. The Control Board includes a Serial-Deserializer interfaceto the LCD+Touchscreen electronics module 2110 and to the OpticalDetection Unit electronics module 2108. The Control Board also includesmodule connectors.

In the exemplary embodiment, the optical detection unit electronicsmodule 2108 contains a dedicated processor. The optical detection unit2108 contains a serializer-deserializer interface. The optical detectionunit 2108 contains LED drivers. The optical detection unit also containshigh gain-low noise photodiode amplifiers. The optical detection unitpreferably has power monitoring capability. The optical detection unitis remotely reprogrammable.

The Heater Board electronics module 2104 is preferably a glass heaterboard. The Heater Board has PCB with bonding pads for glass heater boardand high density connectors.

In the exemplary embodiment, the heater mux (‘multiplex’) boardelectronics module 2106 has 24 high-speed ADC, 24 precision currentsources, and 96 optically isolated current drivers for heating. Theheater mux board has the ability to time-multiplex heating/measurement.The heater mux board has multiplexer banks to multiplex inputs to ADC,and to multiplex current source outputs. The heater mux board has a FPGAwith a soft processor core and SDRAM. The heater mux board has a PowerMonitor with a dedicated processor. The Heater Mux Board is preferablyremotely reprogrammable.

Certain software can be executed in each electronics module. The ControlBoard Electronics Module executes, for example, Control Board PowerMonitor software. The Card Engine electronics module executes anoperating system, graphical user interface (GUI) software, an analyzermodule, and an application program interface (api). The OpticalDetection Unit electronics module executes an optics software module.The Heater Mux Board electronics module executes dedicated Heater Muxsoftware, and Heater Mux Power Monitor software. Each of the separateinstances of software can be modular and under a unified control of, forexample, driver software.

The exemplary electronics can use Linux, UNIX, Windows, or MacOS,including any version thereof, as the operating system. The operatingsystem is preferably loaded with drivers for USB, Ethernet, LCD,touchscreen, and removable media devices such as compact flash.Miscellaneous programs for configuring the Ethernet interface, managingUSB connections, and updating via CD-ROM can also be included.

In the embodiment of FIG. 17, the analyzer module is the driver forspecific hardware. The analyzer module provides access to the Heater MuxModule, the Optical Detection Unit, the Control Board Power Monitor, theReal Time Clock, the High Voltage Power Supply, and the LCD backlight.The analyzer module provides firmware programming access to the ControlBoard power monitor, the Optical Detection Unit, and the Heater MuxModule.

The API provides uniform access to the analyzer module driver. The APIis responsible for error trapping, and interrupt handling. The API istypically programmed to be thread safe.

The GUI software can be based on a commercial, off-the-shelf PEGgraphics library. The GUI can use the API to coordinate the self-test ofoptical detection unit and heater assembly. The GUI starts, stops, andmonitors test progress. The GUI can also implement an algorithm toarrive at a diagnosis from fluorescence data. Such an algorithm can relyon numerical analysis principles known in the art. The GUI providesaccess control to the apparatus has and may have an HIS/LIS interface.

The Control Board Power Monitor software monitors power supplies,current and voltage, and signals error in case of a fault.

The Optics Software performs fluorescence detection which is preciselytimed to turn on/off of LED with synchronous digitization of thephotodetector outputs. The Optics Software can also monitor power supplyvoltages. The Optics Software can also have self test ability.

The Heater Mux Module software implements a “protocol player” whichexecutes series of defined “steps” where each “step” can turn on sets ofheaters to implement a desired microfluidic action. The Heater MuxModule software also has self test ability. The Heater Mux Modulesoftware contains a fuzzy logic temperature control algorithm.

The Heater Mux Power Monitor software monitors voltage and currentlevels. The Heater Mux Power Monitor software can participate inself-test, synchronous, monitoring of the current levels while turningon different heaters.

Overview of an Apparatus for Detecting Fluorescence from an Analyte in aMicrofluidic Channel

The present technology relates to a cartridge, complementary apparatus,and related methods for amplifying, and carrying out diagnostic analyseson, nucleotides from biological samples. The technology includes adisposable or reusable microfluidic cartridge containing multiple samplelanes capable of processing samples in parallel as further describedherein, and a reusable apparatus that is configured to selectivelyactuate on-cartridge operations, to detect and analyze the products ofthe PCR amplification in each of the lanes separately, in allsimultaneously, or in groups simultaneously, and, optionally, candisplay the progression of analyses and results thereof on a graphicaluser interface. Such a reusable apparatus is further described in U.S.patent application Ser. No. ______, entitled “Microfluidic System forAmplifying And Detecting Polynucleotides In Parallel” and filed on Nov.14, 2007, and which is incorporated herein by reference in its entirety.

FIG. 18 shows a perspective view of an exemplary apparatus 100consistent with those described herein, as well as various componentsthereof, such as exemplary cartridge 200 that contains multiple samplelanes, and exemplary read head 300 that contains detection apparatus forreading signals from cartridge 200. The apparatus 100 of FIG. 18 is ableto carry out real-time PCR on a number of samples in cartridge 200simultaneously or serially. Preferably the number of samples is 12samples, as illustrated with exemplary cartridge 200, though othernumbers of samples such as 4, 8, 10, 16, 20, 24, 25, 30, 32, 36, 40, and48 are within the scope of the present description. In preferredoperation of the apparatus, a PCR-ready solution containing the sample,and, optionally, one or more analyte-specific reagents (ASR's) isprepared, as further described elsewhere (see, e.g., U.S. patentapplication publication 2006-0166233, incorporated herein by reference),prior to introduction into cartridge 200. An exemplary kit for preparinga PCR-ready sample, the kit comprising buffers, lysis pellets, andaffinity pellets, has been described elsewhere (see, e.g., U.S.provisional patent application Ser. No. 60/859,284).

In some embodiments, an apparatus includes: a receiving bay configuredto selectively receive a microfluidic cartridge as described herein; atleast one heat source thermally coupled to the receiving bay; and aprocessor coupled to the heat source, wherein the heat source isconfigured to selectively heat individual regions of individual samplelanes in the cartridge, and the processor is configured to controlapplication of heat to the individual sample lanes, separately, in allsimultaneously, or in groups simultaneously; at least one detectorconfigured to detect one or more polynucleotides or a probe thereof in asample in one or more of the individual sample lanes, separately orsimultaneously; and a processor coupled to the detector to control thedetector and to receive signals from the detector.

The receiving bay is a portion of the apparatus that is configured toselectively receive the microfluidic cartridge. For example, thereceiving bay and the microfluidic cartridge can be complementary inshape so that the microfluidic cartridge is selectively received in,e.g., a single orientation. The microfluidic cartridge can have aregistration member that fits into a complementary feature of thereceiving bay. The registration member can be, for example, a cut-out onan edge of the cartridge, such as a corner that is cut-off, or one ormore notches that are made on one or more of the sides in a distinctivepattern that prevents a cartridge from being loaded into the bay in morethan one distinct orientation. By selectively receiving the cartridge,the receiving bay can help a user to place the cartridge so that theapparatus can properly operate on the cartridge. The cartridge can bedesigned to be slightly smaller than the dimensions of the receiving bayby approximately 200-300 microns for easy placement and removal of thecartridge.

The receiving bay can also be configured so that various components ofthe apparatus that operate on the microfluidic cartridge (heat sources,detectors, force members, and the like) are positioned to properlyoperate thereon. For example, a contact heat source can be positioned inthe receiving bay such that it can be thermally coupled to one or moredistinct locations on a microfluidic cartridge that is selectivelyreceived in the bay. Alignment of microheaters in the heater module withcorresponding heat-requiring microcomponents (such as valves, pumps,gates, reaction chambers, etc). The microheaters can be designed to beslightly bigger than the heat requiring microfluidic components so thateven though the cartridge may be off-centered from the heater, theindividual components can still function effectively.

The lower surface of the cartridge can have a layer of mechanicallycompliant heat transfer laminate that can enable thermal contact betweenthe microfluidic substrate and the microheater substrate of the heatermodule. A minimal pressure of 1 psi can be employed for reliableoperation of the thermal valves, gates and pumps present in themicrofluidic cartridge.

In various embodiments of the apparatus, the apparatus can furtherinclude a sensor coupled to the processor, the sensor configured tosense whether the microfluidic cartridge is selectively received.

The detector can be, for example, an optical detector as furtherdescribed elsewhere herein. For example, the detector can include alight source that selectively emits light in an absorption band of afluorescent dye, and a light detector that selectively detects light inan emission band of the fluorescent dye, wherein the fluorescent dyecorresponds to a fluorescent polynucleotide probe or a fragment thereof.Alternatively, for example, the optical detector can include abandpass-filtered diode that selectively emits light in the absorptionband of the fluorescent dye and a bandpass filtered photodiode thatselectively detects light in the emission band of the fluorescent dye;or for example, the optical detector can be configured to independentlydetect a plurality of fluorescent dyes having different fluorescentemission spectra, wherein each fluorescent dye corresponds to afluorescent polynucleotide probe or a fragment thereof; or for example,the optical detector can be configured to independently detect aplurality of fluorescent dyes at a plurality of different locations on amicrofluidic cartridge, wherein each fluorescent dye corresponds to afluorescent polynucleotide probe or a fragment thereof in a differentsample. The detector can also be configured to detect the presence orabsence of sample in a PCR reaction chamber in a given sample lane, andto condition initiation of thermocycling upon affirmative detection ofpresence of the sample.

In various embodiments, the apparatus can further include an analysisport. The analysis port can be configured to allow an external sampleanalyzer to analyze a sample in the microfluidic cartridge. For example,the analysis port can be a hole or window in the apparatus which canaccept an optical detection probe that can analyze a sample or progressof PCR in situ in the microfluidic cartridge. In some embodiments, theanalysis port can be configured to direct a sample from the microfluidiccartridge to an external sample analyzer; for example, the analysis portcan include a conduit in fluid communication with the microfluidiccartridge that directs a liquid sample containing an amplifiedpolynucleotide to a chromatography apparatus, an optical spectrometer, amass spectrometer, or the like.

The heat source can be, for example, a heat source such as a resistiveheater or network of resistive heaters, and the like.

In preferred embodiments, the at least one heat source can be a contactheat source selected from a resistive heater (or network thereof), aradiator, a fluidic heat exchanger and a Peltier device. The contactheat source can be configured at the receiving bay to be thermallycoupled to one or more distinct locations of a microfluidic cartridgereceived in the receiving bay, whereby the distinct locations areselectively heated. The contact heat source typically includes aplurality of contact heat sources, each configured at the receiving bayto be independently thermally coupled to a different distinct locationin a microfluidic cartridge received therein, whereby the distinctlocations are independently heated. The contact heat sources can beconfigured to be in direct physical contact with one or more distinctlocations of a microfluidic cartridge received in the bay. In variousembodiments, each contact source heater can be configured to heat adistinct location having an average diameter in 2 dimensions from about1 millimeter (mm) to about 15 mm (typically about 1 mm to about 10 mm),or a distinct location having a surface area of between about 1 mm²about 225 mm² (typically between about 1 mm² and about 100 mm², or insome embodiments between about 5 mm² and about 50 mm²). Variousconfigurations of heat sources are further described in U.S. patentapplication Ser. No. ______, entitled “Heater Unit for MicrofluidicDiagnostic System” and filed on even date herewith.

In various embodiments, the heat source is disposed in a heating modulethat is configured to be removable from the apparatus.

In various embodiments, the apparatus can include a compliant layer atthe contact heat source configured to thermally couple the contact heatsource with at least a portion of a microfluidic cartridge received inthe receiving bay. The compliant layer can have a thickness of betweenabout 0.05 and about 2 millimeters and a Shore hardness of between about25 and about 100.

In various embodiments, the apparatus can further include one or moreforce members configured to apply force to at least a portion of amicrofluidic cartridge received in the receiving bay. The one or moreforce members are configured to apply force to thermally couple the atleast one heat source to at least a portion of the microfluidiccartridge. The application of force is important to ensure consistentthermal contact between the heater wafer and the PCR reactor andmicrovalves in the microfluidic cartridge.

In various embodiments, the apparatus can further include a lid at thereceiving bay, the lid being operable to at least partially excludeambient light from the receiving bay.

The apparatus preferably also includes a processor comprisingmicroprocessor circuitry, in communication with, for example, the inputdevice and a display, that accepts a user's instructions and controlsanalysis of samples.

In various embodiments, the apparatus can further include at least oneinput device coupled to the processor, the input device being selectedfrom the group consisting of a keyboard, a touch-sensitive surface, amicrophone, and a mouse.

In various embodiments, the apparatus can further include at least onesample identifier coupled to the processor, the sample identifier beingselected from an optical scanner such as an optical character reader, abar code reader, or a radio frequency tag reader. For example, thesample identifier can be a handheld bar code reader.

In various embodiments, the apparatus can further include at least onedata storage medium coupled to the processor, the medium selected from ahard disk drive, an optical disk drive, or one or more removable storagemedia such as a CD-R, CD-RW, USB-drive, or flash memory card.

In various embodiments, the apparatus can further include at least oneoutput coupled to the processor, the output being selected from adisplay, a printer, and a speaker, the coupling being either directlythrough a directly dedicated printer cable, or wirelessly, or via anetwork connection.

The apparatus further optionally comprises a display that communicatesinformation to a user of the system. Such information includes but isnot limited to: the current status of the system; progress of PCRthermocycling; and a warning message in case of malfunction of eithersystem or cartridge. The display is preferably used in conjunction withan external input device as elsewhere described herein, through which auser may communicate instructions to apparatus 100. A suitable inputdevice may further comprise a reader of formatted electronic media, suchas, but not limited to, a flash memory card, memory stick, USB-stick,CD, or floppy diskette. An input device may further comprise a securityfeature such as a fingerprint reader, retinal scanner, magnetic stripreader, or bar-code reader, for ensuring that a user of the system is infact authorized to do so, according to pre-loaded identifyingcharacteristics of authorized users. An input device mayadditionally—and simultaneously—function as an output device for writingdata in connection with sample analysis. For example, if an input deviceis a reader of formatted electronic media, it may also be a writer ofsuch media. Data that may be written to such media by such a deviceincludes, but is not limited to, environmental information, such astemperature or humidity, pertaining to an analysis, as well as adiagnostic result, and identifying data for the sample in question.

The apparatus may further include a computer network connection thatpermits extraction of data to a remote location, such as a personalcomputer, personal digital assistant, or network storage device such ascomputer server or disk farm. The network connection can be acommunications interface selected from the group consisting of: a serialconnection, a parallel connection, a wireless network connection, and awired network connection such as an ethernet or cable connection,wherein the communications interface is in communication with at leastthe processor. The computer network connection may utilize, e.g.,ethernet, firewire, or USB connectivity. The apparatus may further beconfigured to permit a user to e-mail results of an analysis directly tosome other party, such as a healthcare provider, or a diagnosticfacility, or a patient.

In various embodiments, there is an associated computer program productincludes computer readable instructions thereon for operating theapparatus and for accepting instructions from a user.

Apparatus 100 may optionally comprise one or more stabilizing feet thatcause the body of the device to be elevated above a surface on whichsystem 100 is disposed, thereby permitting ventilation underneath system100, and also providing a user with an improved ability to lift system100.

In another preferred embodiment (not shown in the FIGs. herein), acartridge and apparatus are configured so that the read-head does notcover the sample inlets, thereby permitting loading of separate sampleswhile other samples are undergoing PCR thermocycling.

EXAMPLES Example 1 Analyzer and Control Circuitry

An Analyzer unit can contain typical hardware/firmware that can beemployed to drive and monitor the operations on the cartridges as wellas software to interpret, communicate and store the results. The unitcurrently weighs about 20 lbs. and is approximately 10″ wide by 16″ deepby 13″ high. Typical components of the Analyzer can include: (a) ControlElectronics (DAQ), (b) Heater/Sensor Module, (c) Fluorescent DetectionModule, (d) Mechanical Fixtures, (e) Software and (f) User Interface(LCD/Touch screen) (g) Peripherals (CD-ROM, USB/Serial/Ethernetcommunication ports, barcode scanner, optional keyboard). An exemplaryembodiment is shown in FIG. 18.

Control electronics can be spread over four different circuit boardassemblies. These include the Main, MUX, LCD, and Detector boards.

MAIN board: Can serve as the hub of the Analyzer control electronics andmanages communication and control of the other various electronicsub-assemblies. The main board can also serve as the electrical andcommunications interface with the external world. An external powersupply (12V DC/10A; UL certified) can be used to power the system. Theunit can communicate via 5 USB ports, a serial port and an Ethernetport. Finally, the main board can incorporate several diagnostic/safetyfeatures to ensure safe and robust operation of the Analyzer.

MUX Board: Upon instruction from the main board, the MUX board canperform all the functions typically used for accurate temperaturecontrol of the heaters and can coordinate the collection of fluorescencedata from the detector board.

LCD Board: Can contain the typical control elements to light up the LCDpanel and interpret the signals from the touch sensitive screen. TheLCD/touch screen combination can serve as a mode of interaction with theuser via a Graphical User Interface.

Detector Board: Can house typical control and processing circuitry thatcan be employed to collect, digitize, filter, and transmit the data fromthe fluorescence detection modules.

Example 2 Detector Integrated in Force Member

This non-limiting example describes pictorially, various embodiments ofa detection system integrated into a force member, in an apparatus forcarrying out diagnostics on microfluidic samples. An exploded view isshown in FIG. 19.

FIG. 20A: The lid of the apparatus can be closed, which can blockambient light from the sample bay, and place an optical detectorcontained in the lid into position with respect to the microfluidiccartridge.

FIG. 20B: The lid of the apparatus can be closed to apply pressure tothe cartridge. Application of minimal pressure on the cartridge: afterthe slider compresses the cartridge, the slider can compress thecompliant label of the cartridge. This can cause the bottom of thecartridge to be pressed down against the surface of the heater unitpresent in the heater module. Springs present in the slider can deliver,for example approximately 50 lb of pressure to generate a minimumpressure, for example 2 psi over the entire cartridge bottom.

Thermal interface: the cartridge bottom can have a layer of mechanicallycompliant heat transfer laminate that can enable thermal contact betweenthe microfluidic substrate and the microheater substrate of the heatermodule. A minimal pressure of 1 psi can be employed for reliableoperation of the thermal valves, gate and pumps present in themicrofluidic cartridge.

Mechanicals and assembly: the Analyzer can have a simple mechanicalframe to hold the various modules in alignment. The optics module can beplaced in rails for easy opening and placement of cartridges in theAnalyzer and error-free alignment of the optics upon closing. Theheater/sensor module can be also placed on rails or similar guidingmembers for easy removal and insertion of the assembly.

Slider: the slider of the Analyzer can house the optical detectionsystem as well as the mechanical assembly that can enables the opticsjig to press down on the cartridge when the handle of the slider isturned down onto the analyzer. The optics jig can be suspended from thecase of the slider at 4 points. Upon closing the slider and turning thehandle of the analyzer down, 4 cams can turn to push down a plate thatpresses on 4 springs. On compression, the springs can deliverapproximately 50 lb on the optical block. See FIGS. 21A-21C.

The bottom surface of the optics block can be made flat to within 100microns, typically within 25 microns, and this flat surface can pressupon the compliant (shore hardness approximately 50-70) label(approximately 1.5 mm thick under no compression) of the cartridgemaking the pressure more or less uniform over the cartridge. An optionallock-in mechanism can also be incorporated to prevent the slider frombeing accidentally knocked-off while in use.

FIG. 22A shows a side view of a lever assembly 1200, with lever 1210,gear unit 1212, and force member 1214. Assembly 1200 can be used toclose the lid of the apparatus and (through force members 1214) applyforce to a microfluidic cartridge 1216 in the sample well 1217. Oneforce member is visible in this cut away view, but any number, forexample 4, can be used. The force members can be, for example, a manualspring loaded actuator as shown, an automatic mechanical actuator, amaterial with sufficient mechanical compliance and stiffness (e.g., ahard elastomeric plug), and the like. The force applied to themicrofluidic cartridge 1216 can result in a pressure at the surface ofthe microfluidic cartridge 1216 of at least about 0.7 psi to about 7 psi(between about 5 and about 50 kilopascals), or in some embodiments about2 psi (about 14 kilopascals.

FIG. 22B shows a side view of lever assembly 1200, with microfluidiccartridge 1216 in the sample well 1217. A heat source 1219 (for example,a xenon bulb as shown) can function as a radiant heat source directed ata sample inlet reservoir 1218, where the heat can lyse cells inreservoir 1218. A thermally conductive, mechanically compliant layer1222 can lie at an interface between microfluidic cartridge 1216 andthermal stage 1224. Typically, microfluidic cartridge 1216 and thermalstage 1224 can be planar at their respective interface surfaces, e.g.,planar within about 100 microns, or more typically within about 25microns. Layer 1222 can improve thermal coupling between microfluidiccartridge 1216 and thermal stage 1224. Optical detector elements 1220can be directed at the top surface of microfluidic cartridge 1216.

FIGS. 22C and 22D show further cross-sectional views.

Example 3 Exemplary Optics Assembly

In an exemplary embodiment, an assembly comprising a detector on anoptical chassis and a force member that can exert pressure is housed ina plastic enclosure (slider) that can be positioned to cover amulti-lane microfluidic cartridge. The slider has a handle that can beeasily grasped (between 4″ and 5″ width) by a user and drawn towards thefront of the instrument using less than 11 pounds of force. The slideris guided for smooth and easy pushing and pulling with a handle, whichalso serves as a pressure-locking device. The slider's horizontalposition is sensed in both the all-open (fully away from the user) andin the all-forward position, and reported to controlling software. Onceproperly located over a microfluidic cartridge, the slider will belocked in a “down” pressured position, and the user will be required toapply no more than seven pounds of upward force normal to the handle torelease the pressure. Accidental unlocking of the slider mechanism isthereby prevented. The slider and optical chassis pressure assemblyregisters with a heater cassette module positioned underneath amicrofluidic cartridge to within 0.010″. A close fit is important forproper heater/cartridge interface connections.

The slider aligns with the control chip on the heater unit when it is inthe full back position. The height is the same as the distance betweenthe read head bottom and the read area on the cartridge. The slider doesnot come in contact with the control chip but it is positioned such thatthe center of the control chip is in the focal plane of the optic system(0.005″). The slider assembly does not degrade in performance over alife of 10,000 cycles, where a cycle is defined as: beginning with theslider in the back position, and sliding forward then locking the handledown on a cartridge, unlocking the handle and returning it to theoriginal back position. All optical path parts should be non-reflective(anodized, painted, molded, etc.) and do not lose this feature for10,000 cycles. The optics unit is unaffected by a light intensity of<=9,000 foot-candles from a source placed 12″ from the instrument atangles where light penetration is most likely to occur. No degradationof performance is measured at the photo-detector after 10,000 cycles.

A single channel is made that houses two LED sources (blue and amber)and two additional channels that will house one photodiode detector each(four total bored holes). The two paired channels (source and detector)are oriented 43° from each other, measured from the optical axis and arein-line with the other paired channels that are at the same 43°orientation. The holes bored in the optical chassis contain filters andlenses with appropriate spacers, the specifications of which are furtherdescribed herein. The LEDs are held in place to prevent movement as themechanical alignment is important for good source illumination. TheLED's are preferably twisted until the two “hot spots” are aligned withthe reading channels on the cartridge. This position must be maintaineduntil the LED's cannot be moved.

The optical chassis is made of aluminum and is black anodized. Thebottom pressure surface of the optical chassis is flat to +0.001″ acrossthe entire surface. The optical chassis is center-balanced such that thecenter of the optical chassis force is close to the center of thereagent cartridge. The pressure assembly (bottom of the optical chassis)provides uniform pressure of a minimum of 1 psi across all heatersections of the reagent cartridge. The optical assembly can be movedaway from the reagent cartridge area for cartridge removal andplacement. Appropriate grounding of the optical chassis is preferred toprevent spurious signals to emanate to the optic PCB.

The LED light sources (amber and blue) are incident on a microfluidiccartridge through a band pass filter and a focusing lens. These LEDlight sources have a minimum output of 2800 millicandles (blue) and 5600millicandles (Green), and the center wavelengths are 470 (blue) and 575(amber) nanometers, with a half band width of no more than 75nanometers.

The LED light excites at least one fluorescent molecule (initiallyattached to an oligonucleotide probe) in a single chamber on acartridge, causing it to fluoresce. This fluorescence will normally beefficiently blocked by a closely spaced quencher molecule. DNAamplification via TAQ enzyme will separate the fluorescent and quenchingmolecules from the oligonucleotide probe, disabling the quenching. DNAamplification will only occur if the probe's target molecule (a DNAsequence) is present in the sample chamber. Fluorescence occurs when acertain wavelength strikes the target molecule. The emitted light is notthe same as the incident light. Blue incident light is blocked from thedetector by the green only emission filter. Green incident lightsimilarly is blocked from the detector by the yellow emission filter.The fluorescent light is captured and travels via a pathway into afocusing lens, through a filter and onto a very sensitive photodiode.The amount of light detected increases as the amount of the DNAamplification increases. The signal will vary with fluorescent dye used,but background noise should be less than 1 mV peak-to-peak. Thephoto-detector, which can be permanently mounted to the optical chassisin a fixed position, should be stable for 5 years or 10,000 cycles, andshould be sensitive to extremely low light levels, and have a dark valueof no more than 60 mV. Additionally, the photo-detector must becommercially available for at least 10 years. The lenses arePlano-convex (6 mm detector, and 12 mm source focal length) with theflat side toward the test cartridge on both lenses. The filters shouldremain stable over normal operating humidity and temperature ranges.

The filters, e.g., supplied by Omega Optical (Brattleboro, Vt. 05301),are a substrate of optical glass with a surface quality of F/F perMil-C-48497A. The individual filters have a diameter of 6.0±0.1 mm, athickness of 6.0±0.1 mm, and the AOI and ½ cone AOI is 0 degrees and ±8degrees, respectively. The clear aperture is >/=4 mm diameter and theedge treatment is blackened prior to mounting in a black, anodized metalring.

The FITC exciter filter is supplied by, e.g., Omega Optical (PN481AF30-RED-EXC). They have a cut-off frequency of 466±4 nm and a cut-onfrequency of 496±4 nm. Transmission is >/=65% peak and blockingis: >/=OD8 in theory from 503 to 580 nm, >/=OD5 from 501-650 nm, >/=OD4avg. over 651-1000 nm, and >/=OD4 UV-439 nm.

The FITC emitter filter is supplied by, e.g., Omega Optical (PN534AF40-RED-EM). They have a cut-off frequency of 514±2 nm and a cut-onfrequency of 554±4 nm. Transmission is >/=70% peak and blockingis: >/=OD8 in theory from 400 to 504 nm, >/=OD5 UV-507 nm, and >/=OD4avg. 593-765 nm.

The amber exciter filters are supplied by, e.g., Omega Optical (PN582AF25-RED-EXC). They have a cut-off frequency of 594±5 nm and a cut-onfrequency of 569±5 nm. Transmission is >/=70% peak and blockingis: >/=OD8 in theory from 600 to 700 nm, >/=OD5 600-900 nm, and >/=OD4UV-548 nm.

The amber emitter filters are supplied by, e.g., Omega Optical (PN627AF30-RED-EM). They have a cut-off frequency of 642±5 nm and a cut-onfrequency of 612±5 nm. Transmission is >/=70% peak and blockingis: >/=OD8 in theory from 550 to 600 nm, >/=OD5 UV-605 nm, and >/=OD5avg. 667-900 nm.

The spacers should be inert and temperature stable throughout the entireoperating range and should maintain the filters in strict position andalignment. The epoxy used should have optically black and opaquematerial and dry solid with no tacky residue. Additionally, it shouldhave temperature and moisture stability, exert no pressure on the heldcomponents, and should mount the PCB in such a way that it is fixed andstable with no chances of rotation or vertical height changes. 50% ofillumination shall fall on the sample plane within an area 0.1″ (2.5 mm)wide by 0.3″ (7.5 mm) along axis of the detection channel. Fluorescenceof the control chip should not change more than 0.5% of the measuredsignal per 0.001″ of height though a region ±0.010 from the nominalheight of the control chip.

Example 4 Exemplary Optics Board

An exemplary optics board is shown schematically in FIG. 23, and is usedto collect and amplify the fluorescent signature of a successfulchemical reaction on a micro-fluidic cartridge, and control theintensity of LED's using pulse-width modulation (PWM) to illuminate thecartridge sample over up to four channels, each with two color options.Additionally, it receives instructions and sends results data back overan LVDS (low-voltage differential signaling) SPI (serial peripheralinterface). In some embodiments there is a separate instance of thiscircuitry for each PCR channel that is monitored.

The power board systems include: a +12V input; and +3.3V, +3.6V, +5V,and −5V outputs, configured as follows: the +3.3V output contains alinear regulator, is used to power the LVDS interface, should maintain a+1-5% accuracy, and supply an output current of 0.35 A; the +3.6V outputcontains a linear regulator, is used to power the MSP430, shouldmaintain a +/−5% accuracy, and supply an output current of 0.35 A; the+5V output contains a linear regulator, is used to power the plus railfor op-amps, should maintain a +/−5% accuracy, and supply an outputcurrent of 0.35 A; the −5V output receives its power from the +5Vsupply, has a mV reference, is used to power the minus rail for op-ampsand for the photo-detector bias, should maintain a +/−11% voltageaccuracy, and supply an output current of 6.25 mA+/−10%. Additionally,the power board has an 80 ohm source resistance, and the main boardsoftware can enable/disable the regulator outputs.

The main board interface uses a single channel of the LVDS standard tocommunicate between boards. This takes place using SPI signaling overthe LVDS interface which is connected to the main SPI port of thecontrol processor. The interface also contains a serial port forin-system programming.

The optical detection system of FIG. 23 comprises a control processor,LED drivers, and a photo-detection system. In the exemplary embodiment,the control processor is a TI MSP430F1611 consisting of a dual SPI (onefor main board interface, and one for ADC interface) and extended SRAMfor data storage. It has the functions of power monitoring, PWM LEDcontrol, and SPI linking to the ADC and main board. The LED driverscontain NPN transistor switches, are connected to the PWM outputs of thecontrol processor, can sink 10 mA @ 12V per LED (80 mA total), and aresingle channel with 2 LEDs (one of each color) connected to each. Thephoto-detection system has two channels and consists of aphoto-detector, high-sensitivity photo-diode detector, high gain currentto voltage converter, unity gain voltage inverting amplifier, and anADC. Additionally it contains a 16 channel Sigma-delta (only utilizingthe first 8 channels) which is connected to the second SPI port of thecontrol processor.

During assembly of the various components on to the PC board, such asmay occur on a production line, there are the following considerations.The extremely high impedance of the photo-detection circuit means that arigorous cleaning procedure must be employed. Such a procedure mayinclude, for example: After surface mount components are installed, theboards are washed on a Weskleen and blow dried upon exiting conveyor.The belt speed can be set at 20-30. The boards are soaked in an alcoholbath for approximately 3 minutes, then their entire top and bottomsurfaces are scrubbed using a clean, soft bristle brush. The boards arebaked in a 105° F. (40° C.) oven for 30 minutes to dry out allcomponents.

After all the components are installed: the soldered areas of the boardscan be hand wash using deionized water and a soft bristle brush. Thesame soldered areas can be hand washed using alcohol and a soft bristlebrush. The boards are allowed to air dry. Once the board is cleaned, theoptical circuitry must be conformal coated to keep contaminates out.

Example 5 Fluorescence Detection Module

A miniaturized, highly sensitive fluorescence detection system (see FIG.24) can be incorporated for monitoring fluorescence from the biochemicalreactions. This optics module can employ light emitting diodes (LED's),photodiodes and filters/lenses for monitoring, in real-time, thefluorescent signal emanating from the microfluidic cartridge. Thecurrent example module contains six identical detection elements andeach element can be capable of dual-color detection of a pre-determinedset of fluorescent probes.

Software: The software can include two broad parts—user interface anddevice firmware. The user interface software can allow for aspects ofinteraction with the user such as—entering patient/sample information,monitoring test progress, error warnings, printing test results,uploading of results to databases and updating software. The devicefirmware can be the low level software that actually runs the test. Thefirmware can have a generic portion that can be test independent and aportion specific to the test being performed. The test specific portion(“protocol”) can specify the microfluidic operations and their order toaccomplish the test.

FIG. 25 shows screen captures from the programming interface and realtime heat sensor and optical detector monitoring. This real time deviceperformance monitoring is for testing purposes; not visible to the userin the final configuration.

Example 6 Scanning Detector Unit

In one embodiment a detector is configured to scan over multiple lanesof a microfluidic substrate such as in a microfluidic cartridge, ratherthan remain stationary and require a separate detector instancededicated to each lane. It is also an aspect of this embodiment thatmultiple detector units are stacked adjacent to one another therebypermitting simultaneous detection from multiple lanes, even as thedetector is travelling over the microfluidic substrate. It is a furtheraspect of this embodiment that each detector unit is a 4-color system,or is a 1-color system, or is a 2-color system.

FIG. 26 shows a cross-section of the detector. The reader includes anoptical detection unit that can be pressed against a 24-lanemicrofluidic cartridge to optically interface with the PCR lanes as wellas press the cartridge against a microfluidic heater substrate. Thebottom of the optics block has 24 apertures (two rows of 12 apertures)that are similar in dimension to the PCR reactors in the cartridge. Theaperture plate is made of low fluorescent material, such as anodizedblack aluminum and during operation, minimizes the total backgroundfluorescence while maximizing the collection of fluorescent only fromthe PCR reactor (FIG. 27). The bottom of the aperture plate has twobeveled edges that help align two edges of the cartridges appropriatelysuch that the apertures line up with the PCR reactors (FIGS. 28A, 28B).

The optical detection blocks, FIGS. 29 and 30, (total of 8 detectionunits in this example) are assembled and mounted onto a sliding railinside the optical box so that the optical units can be scanned over theapertures (FIG. 29). Each unit is able to excite and focus a certainwavelength of light onto the PCR reactor and collect emittedfluorescence of particular wavelength into a photodetector. Each blockof the embodiment shown has 2 units and is configured to measure aparticular frequency of light from 2 separate locations, such as where amicrofluidic substrate is configured with 2 banks (top and bottom) ofPCR reactors. By using 4 different colors, one in each of the fourparallel detection units, on the top 4 channels and repeating the 4colors in the bottom channels, the entire scanner can scan up to 4colors from each of the PCR lanes.

The optics block can be machined out of aluminum and anodized orinjection molded using low fluorescence black plastic (FIG. 30).Injection molding can dramatically reduce the cost per unit and alsomake the assembly of optics easier. The designed units can be stackedback-to-back.

The foregoing description is intended to illustrate various aspects ofthe present technology. It is not intended that the examples presentedherein limit the scope of the present technology. The technology nowbeing fully described, it will be apparent to one of ordinary skill inthe art that many changes and modifications can be made thereto withoutdeparting from the spirit or scope of the appended claims.

1. A diagnostic apparatus, comprising: one or more microfluidic channelsconfigured to amplify one or more polynucleotides; and one or morefluorescence detectors configured to detect presence of the one or morepolynucleotides in the one or more channels by detecting fluorescentlight emitted from a probe associated with the one or morepolynucleotides, wherein the one or more detectors each comprise: afirst LED emitting light of a first color; a second LED emitting lightof a second color; a first photodiode configured to collect emittedlight of the first color; a second photodiode configured to collectemitted light of the second color; and wherein the first and secondphotodiodes are each connected to a pre-amplifier circuit having atime-constant of less than of about 1 second.
 2. The apparatus of claim1, wherein the time constant is 50-100 ms.
 3. The apparatus of claim 1,wherein the pre-amplifier circuit further comprises a resistor having aresistance in excess of 0.5 GΩ.
 4. The apparatus of claim 1 wherein theone or more microfluidic channels is in a removable microfluidiccartridge disposed within a receiving bay in the apparatus.
 5. Theapparatus of claim 1, further comprising a processor coupled to the oneor more detectors and configured to receive an amplified signal from theone or more detectors and transmit a diagnostic result to a user.
 6. Theapparatus of claim 1, wherein the one or more detectors comprise abandpass-filtered diode that selectively emits light in the absorptionband of the probe and a bandpass filtered photodiode that selectivelydetects light in the emission band of the probe.
 7. The apparatus ofclaim 1, wherein the one or more detectors are configured toindependently detect a plurality of probes at a plurality of differentlocations, wherein each probe corresponds to a polynucleotide probe or afragment thereof.
 8. The apparatus of claim 1, further comprising acontact heat source configured to be thermally coupled to the one ormore microfluidic channels, whereby the one or more microfluidicchannels are selectively heated to amplify the one or morepolynucleotides therein.
 9. The apparatus of claim 4, further comprisinga lid at the receiving bay, the lid being operable to at least partiallyexclude ambient light from the receiving bay.
 10. The apparatus of claim9, wherein the one or more detectors are housed in the lid.
 11. Theapparatus of claim 1, wherein an aperture is disposed between each ofthe one or more detectors and the respective one or more microfluidicchannels.
 12. The apparatus of claim 1, wherein the first and secondLEDs and the first and second photodetectors are assembled onto a singleoptics housing, positioned on the same side relative to the microfluidiccartridge.
 13. The apparatus of claim 1, wherein the one or morefluorescence detectors are mounted on a movable assembly that permitsthe one or more detectors to scan across the one or more channels.
 14. Adiagnostic apparatus, comprising: one or more microfluidic channelsconfigured to amplify one or more polynucleotides; and one or morefluorescence detectors configured to detect presence of the one or morepolynucleotides in the one or more channels by detecting fluorescentlight emitted from a probe associated with the one or morepolynucleotides, wherein the one or more detectors each comprise: a LEDemitting light of a specified color that excites the probe; a photodiodeconfigured to collect emitted light of the specified color; and whereinthe photodiode is connected to a pre-amplifier circuit having atime-constant of less than about 1 s.
 15. A diagnostic apparatus,comprising: one or more microfluidic channels configured to amplify oneor more polynucleotides; and one or more fluorescence detectorsconfigured to detect presence of the one or more polynucleotides in theone or more channels by detecting fluorescent light emitted from a probeassociated with the one or more polynucleotides, wherein the one or moredetectors each comprise: a first LED emitting light of a first color; asecond LED emitting light of a second color; a first photodiodeconfigured to collect emitted light of the first color; a secondphotodiode configured to collect emitted light of the second color; andwherein the first and second photodiodes are each connected to apre-amplifier circuit having a Gain of about 10⁹.
 16. A fluorescentdetector, comprising: a LED emitting light of a specified color thatexcites a probe associated with one or more polynucleotides containedwithin a microfluidic channel; and a photodiode configured to collectemitted light of the specified color, wherein the photodiode isconnected to a pre-amplifier circuit having a time-constant of less thanabout 1 s.